Cystic fibrosis (CF) is one of the most common genetic diseases among Caucasians and is a contributing factor in causing suffering among children and adults. CF affects the mucus-producing glands and other exocrine glands in the body. The gene encoding CFTR, the absence of function of which is responsible for causing CF, is located on chromosome 7q. Lack of CFTR function causes abnormal mucus production in the respiratory and gastrointestinal tracts and abnormal sweat gland function. Clinically, CF is characterized by chronic respiratory infections and obstructive lung disease, pancreatic gland insufficiency leading to an inability to digest fats, male infertility and abnormally high levels of electrolytes in the sweat.
CF is the most common lethal autosomal recessive disorder in the Caucasian population occurring in approximately 1 in 2,500 live births. Therefore, about 1 in 50 people is a carrier of CF. It is less common in other populations, occurring in approximately 1 in 18,000 African-Americans and about 1 in every 33,000 Asian-Americans. The median age of survival is 31. The life expectancy of those with cystic fibrosis (CF) is increasing due to the availability of new medications, rigorous care and prompt diagnosis of the disease.
The sweat gland defect in CF has been well characterized for years and is presently used to diagnose CF by the use of what is called the "pilocarpine iontophoresis sweat test."
Sweat production is stimulated through two pathways: the cholinergic pathway and by the adrenergic/sympathetic pathway (i.e., fight or flight response). Thus, sweat production can be stimulated by both cholinergic and adrenergic agonists. Collection of cholinergic stimulated sweat, using the cholinergic agonist pilocarpine, and the subsequent measurement of sweat chloride concentration is the basis of a standard diagnostic test for cystic fibrosis (CF), the pilocarpine iontophoresis sweat test. Stimulation of sweat production with pilocarpine leads to initial production of an isotonic secretion in the sweat gland. In non-CF patients, as the secretion traverses the sweat duct, chloride is reabsorbed. This leads to low concentration of chloride in sweat as it appears on the skin. This chloride resorption is dependent on the presence of functional CFTR. In CF patients, who lack functional CFTR, the sweat chloride concentration remains high, and distinguishes most, but not all, CF from non-CF patients. However, this technique does not distinguish heterozygote carriers of CFTR mutations from non-carriers, nor does the sweat chloride concentration correlate with disease severity. Furthermore, the pilocarpine iontophoresis sweat test involves the use of a sweat test apparatus consisting of electrodes and a voltage source and the use of specially trained personnel. These methods require the elution of the sweat electrolytes collected on the pads and determination of chloride content of the sweat. While this method remains the "gold standard", it occasionally yields ambiguous results. Therefore, it would be useful to have an alternative method of diagnosing CF.
As discussed above, sweat production is also stimulated by .beta.-adrenergic agonists that lead to increases in intracellular cyclic AMP (cAMP) in the sweat duct epithelia. This response is dependent on the presence of functional CFTR, which is a cAMP-stimulated chloride transporter. It has been reported that CF subjects do not sweat after .beta.-adrenergic stimulation. (Sato, K., et al., (1984), J. Clin. Invest., 73:1763-1771.) It has also been reported that non-CF subjects have markedly increased sweat production rates compared to CF subjects, (Sato, K., et al. (1984), supra.; Sato, K., et al., (1988), J. Lab. Clin. Med., 111:511-518) while non-affected carriers of a CFTR mutation have intermediate sweat rates. (Sato K. et al. (1988), supra; Behm et al., Pediatric Research, Vol. 22, No. 3 (1987), pp. 271-276). The rate of sweat production in response to .beta. agonists is proportional to the number of functional copies of CFTR present in the subject's genotype.
It has also been reported that there are gender-related differences in cAMP-stimulated sweat rates due to sweat gland density, (Sato, et al, 1988 supra). Thus, it would be useful to have a method of determining sweat rates that is not gender-dependent.
The previously described method of measuring sweat rate in response to .beta.-adrenergic stimulation is described in Sato, K., et al. (1984), supra.; and Sato K. et al. (1988), supra. Briefly, the method used by Sato, et al. involves the collection of beads of sweat secreted into an oil-filled sweat collection ring which is glued to the skin. The sweat collection chamber comprises a Teflon ring with a 7-mm hole in the center. This ring is glued to the skin of the forearm with contact cement. Paraffin oil, saturated with water, is poured into the trough of the chamber. A solution containing isoproterenol is injected into the dermis under the center of the test site. The sweat beads are collected using a glass capillary under a stereomicroscope. Sweat rates are calibrated by transferring the sweat sample into a constant bore calibration pipette. The procedure is depicted in Figure 2 of Sato, K., et al. (1984), supra. Sato determines the nanoliters of sweat produced over time, i.e., the sweat rate. See also Behm et al., Pediatric Research, Vol. 22, No. 3 (1987), pp. 271-276.
Other methods of measuring isoproterenol-stimulated sweat rate involve the use of a water vapor analyzer. Sato, et al, (1988) supra. .beta.-adrenergic sweating is induced by injecting a solution containing isoproterenol to form an intradermal wheal. A capsule rim is coated with silicone grease, placed on the wheal and secured with tape. Dry nitrogen gas is introduced into the capsule and moisture in the outflow nitrogen gas is monitored by an electrolytic water vapor analyzer. The analyzer requires calibration and the leakage of nitrogen gas from the capsule-skin junction needs to be minimized.
The sweat rate of Sato is difficult to determine due to small amount of sweat that is produced. As a result, the sweat rate test is used in only a few CF centers and is not yet routinely recommended.
It would be useful to have the ability to quantitatively measure cAMP-induced sweat rates to improve the detection of CF patients and heterozygous CF carriers. It would also be useful to have a mechanism to study the effects of new and potentially useful systemic CFTR repair therapies for CF. It would especially be useful to have a mechanism to study systemic therapies that produce partial, rather than full, correction of the CFTR defect.
Presently used methods for stimulating sweat use pilocarpine iontophoresis and measure the amount of chloride produced in the sweat. These devices measure sweat produced by the cholinergic pathway, as is discussed above, and do not reliably distinguish between heterozygous carriers of the diseases and those who have CF.
It would be useful to have a method for measuring the amount of sweat produced by the .beta.-adrenergic pathway that is less laborious and less technically demanding than the presently known methods.